Although most patients with lung cancer are smokers, only a minority of smokers actually develop lung cancer. Considerable evidence suggests that genetic factors play an important role in determining which smokers develop lung cancer. The overall goal of this project is to define the genetic determinants for individual susceptibility to epithelial cell DNA damage in smokers, which underlie the genesis of lung cancer. In preliminary studies of patients with lung cancer, we have sampled epithelial cells from several sites commonly involved in smoking related cancer. These studies have demonstrated wide inter- subject variations in the frequency of loss of heterozygozity (LOH), an indicator of tobacco-smoke induced DNA damage, and frequent loss of the same allele in epithelial cells from the airways, buccal and bladder samples. We will confirm these results in a larger group of patients by examining LOH in two populations of smokers who are genetically at increased risk for developing lung cancer; subjects with chronic obstructive lung disease and their smoking siblings, and lung cancer patients less than 60 years of age and their smoking siblings. We will correlate the frequency of LOH, chromosomal sites of LOH, and alleles lost in buccal and bladder epithelial DNA obtained from probands and their smoking siblings. Since the genomic DNA is obtained from buccal scrapings and urine samples, it allows us to sample a large number of smokers in a non-invasive fashion. Furthermore, we have already validated the sensitivity of measuring LOH in our high throughput method using fluorescent labeled markers with serial dilution of the DNA and radiolabeled markers. We would correlate observations made in patient tissue samples with inducible effects in a surrogate model system. To define potential molecular mechanisms responsible for frequency and sites of LOH, we will measure LOH in lymphocytes passaged for several generations from the same subjects after exposure to tobacco carcinogens in vitro. We will determine whether the amounts of LOH correlate with DNA repair proficiency and specific alleles lost represent susceptible DNA sequences that are inherited as well as the role of DNA methylation patterns induced or imprinted in determining the allele(s) involved in LOH. We will substantiate the in vitro data with epithelial cells from clinical samples. We postulate that smoking siblings will display a high degree of concordance for amounts, sites and alleles lost and that may relate to the heritable factors being explored in this proposal. We believe that the findings from these studies will enable the localization of hot spots for genetic alterations on the genome and hence accelerate the identification of genes targeted for inactivation during tobacco smoke derived carcinogen induced lung cancer. In the long-term, these target genes may serve as nodal points for therapeutic intervention, diagnosis, prognosis and management of the disease.